Regulation of acid-base homeostasis (blood pH ~7.4) and Na+ homeostasis is critical for animal life. In all vertebrates, including mammals, homeostasis is achieved through strict regulation of levels of NaHCO3. The electrogenic Na+ bicarbonate cotransporter, NBCel, is a major regulator of NaHCO3 levels through its transport activity at the basolateral membrane of the renal proximal tubule. The importance of this transporter is shown by naturally occurring, recessive, point mutations (R298S , R510H, S427L) in human kidney NBCel (hkNBCel), which cause profound proximal renal tubular acidosis (pRTA), glaucoma and cataracts. Blood pH <7.1 and [HCO3-] <11 mM in these patients indicate that hkNBCel is THE major HC03 absorption path of the kidney. However, the mechanism by which NBCel inactivation leads to pRTA and ocular pathologies is unclear. Our preliminary experiments with hkNBCel mutations show that ion affinities are localized to discrete areas of the NBCel protein. Biophysical analysis and uncompensated pRTA (the mutant NBCel phenotype), indicate that kNBCel has a major role in respiratory compensation as well as renal transport. Because of the compelling phenotype of patients with single amino acid mutations in kNBCel, we propose using kNBCel for biophysical experiments designed to reveal regions of the protein responsible for components of its function. Evidence from human mutations shows definitively that single amino acid changes in hkNBCel drastically alter its activity. We hypothesize that examination of the function of hkNBCel bearing additional sequence modifications will identify critical subdomains responsible for its function. This information can be used to design therapeutic agents targeted to those subdomains to modify the activity of this critical transporter to treat metabolic acidosis, glaucoma and cataracts. To investigate this hypothesis we will pursue 3 aims. First, we will functionally test our structural model by evaluating biophysical properties of mutations in the N-terminus, new human NBCel mutations and the role of NBCel dimers. Second, we will determine the functional roles of the isoform specific N-termini of NBCel. Third, we will use chimeras of human kNBCel with fugu-NBCel to delimit ion binding and/or permeation paths via the NBCel transmembrane domain of the protein. Lay Public Health statement: NBCel is the protein in the kidney responsible for absorbing sodium bicarbonate (baking soda). Human NBCel mutations cause severe kidney disease (metabolic acidosis) and eye disease (glaucoma and cataracts). NBCel from a salt-water puffer fish has some dramatic functional differences though the protein is only slightly different from human NBCel. We will use these NBCel mutations and human/fish differences to determine how this protein causes kidney and eye disease and how to modify its activity to prevent or treat disease.